INTRODUCTION
Scorpion stings represent an important and serious public health problem in tropical and subtropical countries (Gueron et al 2000). Although Hemiscorpious lepturusis one of the highly distributed medically important scorpion in south west part of Iran, but the majority of stings that occur in through our
*Author for correspondence.Email: A.zare@rvsri.ir
country are attributed to the Buthidae family, which includes Mesobuthus eupeus, Androctonus crassicauda, Odontobuthus odonturus and Apistobuthus pertigus (Radmanesh 1990). Scorpion (Me)is one of the major scorpions in Iran and its stings constitute a public health problem In addition, Mesobuthus eupeusshows high geographic distribution in Turkey, Iraq, Turkistan, Afghanistan and Pakistan.
Therefore, Me is known as a central Asian scorpion
Full Article
Biochemical and Histopathological study of Mesobuthus eupeus scorpion venom in the experimental rabbits
Zayerzadeh1, E., Zare Mirakabadi*2, A., Koohi3, M.K.
1. Department of Anaerobic bacterial vaccine research & production, Razi vaccine and serum research institute, Karaj, Iran
2. Department of Venomous Animals and antivenom Production, Razi vaccine and serum research institute, Karaj, Iran
3. Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Received 02 May 2011; accepted 20 Aug 2011
ABSTRACT
In tropical and subtropical countries, envenomation by scorpions (so-called scorpionism) represents a serious public health problem. In the present study, the toxic effects of mice LD50 injections ofMesobuthus eupeus (Me) venom on the kidney and liver of anesthetized rabbits were investigated. Six rabbits were selected and ALT, AST, BUN and creatinine were measured at 0, 1 and 3 hours after envenomation and histopathological studies were carried out postmortem. All the animals showed signs and symptoms of envenomation within 30-40 minutes and died 3 to 3.5 hours after venom injection. Histopathological examinations revealed glumerolar congestion, dilated vessels of interstitium and focal interstitial congestion in the kidney and focal hemorrhage, central vein congestion, congested vessels in portal areas and dilated sinusoids in the liver at 3 to 3.5 hrs following venom injection. In addition, biochemical analyses indicated significant rise in the levels of ALT and creatinine followingMesobuthus eupeus envenomation in animals at 3 hrs. However no significant changes were observed at 1 hr. In conclusion, scorpion (Mesobuthus eupeus) venom leads to damage in vital organs such as liver and kidney.
Keywords: Mesobuthus eupeus, rabbit, envenomation, histopathological changes, ALT, creatinine.
(Ozkanet al2008). Radmanesh demonstrated that 45%
of all cases of scorpion stings in Iran were due to Me (Radmanesh 1990). Investigations showed that following a Me sting, victims have presented severe pain, hyperemia, edema, thirst, dry mouth, hypotension, nausea, hypertension, elevated bronchial secretion, difficulty in breathing, tachycardia and cyanosis (Ozcan et al 2005). In addition, there are some reports of the incidence of acute renal failure and hepatic complications following some scorpions of Buthidae and Scorpionidae family (D'Suze et al 2004, Ismailet al1998, Pipelzadeh et al., 2006, Zare et al., 2007, Zare et al., 2006). To the best of our knowledge, no report has been published regarding the hepatic and renal histopathological as well as biochemical effects of Iranian Me scorpion venom. Therefore, the present study was undertaken to investigate hepatic and renal effects in anesthetized rabbits following Me envenomation.
MATERIALS AND METHODS
Venom. Me venom was obtained using electric shock at the scorpion telson in the Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute of Iran. It was lyophilized and stored at 4°C until use.
Experimental Protocol. Six male New Zealand white rabbits with an average weight of 2 ± 0.3 kg were used in this study. All animals were housed under conditions of controlled light (12L: 12D cycle), temperature (24 ± 1°C) and humidity (55 ± 5%), with standard diet and water available ad libitum. All animals were injected with 50 mg/kg ketamine and 5 mg/kg xylazine (intramuscular to induce anesthesia).
All animals were kept in compliance with the recommendations of the Animal Care Committee of the Tehran University based on the Guide for Care and Use of Laboratory Animals (US NIH publication 86-23, revised 1985). All animals were subcutaneously injected with a lethal dose (4.5 mg/kg of body weight) considering LD50 ofMevenom in mice.
Histopathological Analyses. After the animals died the liver and kidneys were removed carefully and immersed in 10% buffered formalin at room temperature and then sectioned transversely into 3-4 μm slices. Specimens were dehydrated in a graded series of alcohol and xylene and embedded in paraffin.
Multiple slices were made and stained by hematoxilin and eosin stains. Sections were viewed and photographed using a Nikon E200® light microscope (Japan).
Biochemical analyses. Blood samples were collected at 0, 1 and 3 hours following venom injection by cephalic vein puncture, in tubes without anticoagulant.
Serum was separated and used for determination of Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine were assayed in the sera using the photometry technology (COBAS INTEGRA® 400 plus system and ALT, AST, BUN, creatinine kits, Roche, Germany) according to the manufacturer's instructions.
The enzyme values were expressed in international units (U/L).
Statistical Analyses. All results were expressed as mean ± SEM. The statistical significance of differences among groups was analyzed by the Student’s t-test.
Data were considered statistically significant if p- values were < 0.05.
RESULTS
Histopathological analyses. Post-mortem examination revealed central vein congestion (figure 1A), congested vessels in portal areas, dilated sinusoids (figure 1B) and steatohepatitis (figure 1C) in liver of animals treated with Me scorpion venom. In addition, histopathological evaluation of the kidneys tissues demonstrated glumerolar congestion characterized by presence of erythrocytes in the glumeroles and interstitial space (figures 1 D, E), dilated vessels of interstitium and focal interstitial congestion (figure 1D).
Metabolic changes. ALT level was significantly increased at 3 h following Me venom injection but there were no differences between control and envenomed animals regarding ALT serum level at 0 h and 1 h after venom injections. In addition, there were no alterations in AST levels in envenomed animals at 0 h, 1 h and 3 h followingMe venom injections (Table 1).
Figure 1. Histopathological changes of liver (A, B, C) and kidney (D, E) tissue induced by Mesobuthus eupeusvenom (90 μg/20 g body weight). From 3 to 3.5 hours after evenomation.
(A) central vein congestion, (B) congested vessels in portal areas and dilated sinusoids, (c) steatohepatitis in liver are present. Three hours after envenomation, (D) glumerolar congestion, dilated vessels of interstitium and focal interstitial congestion (10x) (E) glumerolar congestion (40x) in kidney were present.
Sections were viewed and photographed using a Nikon E200® light microscope (Japan).
Significant creatinine serum elevation was observed at 3 h afterMe venom injection. However, there were no significant differences between control and envenomed
rabbits regarding creatinine serum level at 0 h and 1 h after venom inoculations. Furthermore, there were no significant changes in BUN serum levels in envenomed animals at different time following Me venom inoculations (Table 2).
DISCUSSION
Stings of medically important scorpions from buthidae family scorpions with medical importance produce some disturbances in major organs. In the present study the dose of venom was selected based on LD50 of the venom in mice to observe the acute toxic effects of theMe venom on vital organs. In this study, injection of Me scorpion venom in the rabbits caused histopathological complications such as central vein congestion, congested vessels in portal areas, dilated sinusoids and steatohepatitis. Dَ Suze et al reported aggregation of leukocytes, mainly neutrophils, in the portal vein and hepatocytes with Mallory’s body near to sinusoids in liver of envenomed rams with Tityus discrepans scorpion venom (D'Suze et al 2004). In addition, they have shown that Tityus discrepans venom caused macrophages, neutrophils and lymphocytes infiltration in liver. Another observations were hepatocytes necrosis in zones surrounding portal venules through which leukocyte were infiltrating (D'Suzeet al 2004). On the other hand, in our study, although the rise at 1hr following venom injection was non significant as compared with control group , but at 3 hrs it was found to be significant. ALT is the most important hepatic enzyme that increaseses during hepatic injury or inflammation. These change was similar to those observed in rabbits receiving venom of scorpions of the Buthidae family (Zare et al 1994b, Zareet al1994a). In addition, Zare et al showed ALT increment in envenomed rabbits with Hemiscorpius lepturus scorpion venom (Zare et al 2007). Moreover, Hepatic toxicity has reported in Hemiscorpious lepturus scorpion victims (Pipelzadeh et al 2006).
Pharmacokinetic studies of the distribution of scorpion venom in rats have shown that the concentration of
A B
A
CA D
CA
EA
Table 1.Serum ALT and AST levels in anesthetized rabbits at 1 and 3hrs followingM.e venom injection.
ALT(U/L) AST(U/L)
Time(h) control envenomed P value control envenomed P value
0 26.2 ± 2.6 23.4 ± 3.8 NS 17.4 ± 2.1 19.1 ± 3.5 NS
1 28.4 ± 3.1 35.5 ± 4.1 NS 22.2 ± 4.1 25.1 ± 5.4 NS
3 25.3 ± 4.4 57.2 ± 3.7 S 20.1 ± 5.4 29.5 ± 4.9 NS
Mean values (standard error) are shown for the six animals in each group. NS: Not significant, S: significant
Table 2.Serum Creatinine and BUN levels in anesthetized rabbits at 1 and 3 hrs followingM.evenom injection.
Creatinine(mg/dL) BUN(mg/dL)
control envenomed P value control envenomed P value
0 0.87 ± 0.23 0.79 ± 0.36 NS 24.3 ± 6.1 21.1 ± 9.4 NS
1 1.02 ± 0.34 0.9 ± 0.44 NS 23.7 ± 3.3 24.4 ± 10.8 NS
3 1.07 ± 0.2 1.93 ± 0.31 S 25.4 ± 4.6 32.8 ± 4.2 NS
Mean values (standard error) are shown for the six animals in each group. NS: Not significant, S: significant
toxins in the liver reached their highest level 15 min after inoculation (Nunan et al2003, Nunan et al2004).
Hence, ALT increament and liver complications in the present work confirmed that lethal dose ofMe venom caused alterations in the liver function of rabbits. In addition, although the rise in serum was observed AST at 1 and 3hrs following venom injection, but the rises were non significant .The non significant rise in AST can be due to limitation of number and variation in physiological response of animals. In our study, injection of Me scorpion venom in rabbits caused histopathological complications such as glumerolar congestion, dilated vessels of interstitium and focal interstitial congestion in kidneys of all animals.
Pharmacokinetic studies in rats have previously shown that scorpion venom spreads rapidly from blood to tissues, and especially the kidneys, reaching a maximum concentration around 15 min after inoculation (Ismail et al 1998, Nunan et al 2003, Nunanet al2004). Martins et al have demonstrated that Snake (Crotalus durissus cascavella) venom induced proximal tubular damage in perfused isolated kidney within two hours (Martinset al1998).
With respect to creatinine level (table 1), the significant statistical difference (p<0.05) was observed between groups at 3 h but there was no alteration in BUN level, which done to evaluated renal lesions. Creatinine and BUN are the final products of protein metabolism and their levels will be increased in renal failure. There are some reports of the incidence of acute renal failure, with increased urea and uric acid concentrations, reduced urinary volume and diminished creatinine secretion, in victims that had been stung by Androctonus, Leiurus and Buthus scorpions (Ismail et al1998). Zare et al demonstrated creatinine and BUN increment following Odonthobuthus doriae scorpion venom injection in experimentally rabbits (Zare et al 2006). However, based on the creatinine elevation and kidney complications observed in this study, it is clear that Me venom in the doses used caused alterations in the renal function rabbits. Clinical manifestations of scorpion envenomation are usually complex in nature and can be attributed mainly to noticeable overactivity of the autonomic nervous system similar to pheochromocytoma. Many toxins in scorpion venoms can interfere selective actions on voltage-gated Na+
channels and/or on voltage-gated and other K+
channels that result in the massive release of autonomic neurotransmitters (described as ‘an autonomic storm’) as a major donor to the pathophysiolgy of scorpion envenomation (Gwee et al 2002). Therefore, hepatic and renal injury can be due to massive release of catecholamines and resultant vasoconstriction and hypertention in liver and kidney. However, these lesions may also be caused by action of other substances related to scorpion envenomation such as cytokines and inflammatory mediators (D'Suze et al 2003, Fukahara et al 2003). Furthermore, Me venom may be toxic to the vessel wall. Endothelial cells are the first cellular barrier to blood-borne toxins. These cells are susceptible to toxic effects. Toxins that reach the subendothelial space may cause injury to medial smooth muscle cells and/or adventitial fibroblasts (Klassen & Watkins 2003). Therefore, the direct effect of Me venom on endothelial cells of liver and kidney vessels leading to hepatic and renal injury are to be considered too.
In conclusion,M. eupeus venom administrated at dose of 4.5 mg/kg was able to induce alterations in liver and kidney organs of rabbits. Our results suggest that these complications induced are multi-factorial occurrences induced by the toxins present in the venom.
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